Anming Xiong

Isolation and Transcriptional Profiling of Purified Hepatic Cells Derived from Human Embryonic Stem Cells

The differentiation of human embryonic stem cells (hESCs) into functional hepatocytes provides a powerful in vitro model system for studying the molecular mechanisms governing liver development. Furthermore, a well‐characterized renewable supply of hepatocytes differentiated from hESCs could be used for in vitro assays of drug metabolism and toxicology, screening of potential antiviral agents, and cell‐based therapies to treat liver disease. In this study, we describe a protocol for the differentiation of hESCs toward hepatic cells with complex cellular morphologies. Putative hepatic cells were identified and isolated using a lentiviral vector, containing the α‐fetoprotein promoter driving enhanced green fluorescent protein expression (AFP:eGFP). Whole‐genome transcriptional profiling was performed on triplicate samples of AFP:eGFP+ and AFP:eGFP− cell populations using the recently released Affymetrix Exon Array ST 1.0 (Santa Clara, CA, http://www.affymetrix.com). Statistical analysis of the transcriptional profiles demonstrated that the AFP:eGFP+ population is highly enriched for genes characteristic of hepatic cells. These data provide a unique insight into the complex process of hepatocyte differentiation, point to signaling pathways that may be manipulated to more efficiently direct the differentiation of hESCs toward mature hepatocytes, and identify molecular markers that may be used for further dissection of hepatic cell differentiation from hESCs.

Mechanism of an Amphipathic α-Helical Peptide’s Antiviral Activity Involves Size-Dependent Virus Particle Lysis

The N-terminal region of the hepatitis C virus (HCV) nonstructural protein NS5A contains an amphipathic alpha-helix that is necessary and sufficient for NS5A membrane association. A synthetic peptide (AH) comprising this amphipathic helix is able to lyse lipid vesicles that serve as a model system for virus particles. Based on quartz crystal microbalance-dissipation (QCM-D) experiments, the degree of vesicle rupturing was found to be inversely related to vesicle size, with maximal activity in the size range of several medically important viruses. In order to confirm and further study vesicle rupture, dynamic light scattering (DLS) and atomic force microscopy (AFM) experiments were also performed. The size dependence of vesicle rupturing helps explain the peptides observed effect on the infectivity of a wide range of viruses. Further, in vitro studies demonstrated that AH peptide treatment significantly decreased the infectivity of HCV particles. Thus, the AH peptide might be used to rupture HCV particles extra-corporally (for HCV prevention) and within infected individuals (for HCV therapy).

Transdifferentiation of adipose-derived stem cells into hepatocytes: a new approach.

Background: Several studies have demonstrated techniques in differentiating human adipose‐derived stem cells (hADSCs) into hepatocytes. Unfortunately, transdifferentiation is inefficient, and the function of these induced hepatocyte‐like cells (which we termed ‘iHeps’) is low compared with that of real hepatocytes.

Viral infection of human progenitor and liver-derived cells encapsulated in three-dimensional PEG-based hydrogel

We have studied the encapsulation of human progenitor cells into 3D PEG hydrogels. Replication-incompetent lentivirus promoter reporter vectors were found to efficiently detect the in vivo expression of human hepatic genes in hydrogel-encapsulated liver progenitor cells. Similarly, hydrogel-encapsulated cells could be efficiently infected with hepatitis C virus, and progeny infectious virus could be recovered from the media supernatants of the hydrogels. Provocatively, the diameters of these virus particles range from ∼50 to 100 nm, while the calculated mesh size of the 8 k hydrogel is 44.6 ± 1.7 ˚ A. To reconcile how viral particles can penetrate the hydrogels to infect the encapsulated cells, we propose that microfractures/defects of the hydrogel result in a functional pore size of up to 20 fold greater than predicted by theoretical mesh calculations. These results suggest a new model of hydrogel structure, and have exciting implications for tissue engineering and hepatitis virus studies. M This article features online multimedia enhancements (Some figures in this article are in colour only in the electronic version)

Hydrophobic nanoparticles improve permeability of cell-encapsulating poly(ethylene glycol) hydrogels while maintaining patternability.

Cell encapsulating poly(ethylene glycol) hydrogels represent a promising approach for constructing 3D cultures designed to more closely approximate in vivo tissue environment. Improved strategies are needed, however, to optimally balance hydrogel permeability to support metabolic activities of encapsulated cells, while maintaining patternability to restore key aspects of tissue architecture. Herein, we have developed one such strategy incorporating hydrophobic nanoparticles to partially induce looser cross-linking density at the particle-hydrogel interface. Strikingly, our network design significantly increased hydrogel permeability, while only minimally affecting the matrix mechanical strength or prepolymer viscosity. This structural advantage improved viability and functions of encapsulated cells and permitted micron-scale structures to control over spatial distribution of incorporated cells. We expect that this design strategy holds promise for the development of more advanced artificial tissues that can promote high levels of cell metabolic activity and recapitulate key architectural features.

Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIβ.

Type III phosphatidylinositol 4-kinase (PI4KIIIβ) is an essential enzyme in mediating membrane trafficking and is implicated in a variety of pathogenic processes. It is a key host factor mediating replication of RNA viruses. The design of potent and specific inhibitors of this enzyme will be essential to define its cellular roles and may lead to novel antiviral therapeutics. We previously reported the PI4K inhibitor PIK93, and this compound has defined key functions of PI4KIIIβ. However, this compound showed high cross reactivity with class I and III PI3Ks. Using structure-based drug design, we have designed novel potent and selective (>1000-fold over class I and class III PI3Ks) PI4KIIIβ inhibitors. These compounds showed antiviral activity against hepatitis C virus. The co-crystal structure of PI4KIIIβ bound to one of the most potent compounds reveals the molecular basis of specificity. This work will be vital in the design of novel PI4KIIIβ inhibitors, which may play significant roles as antiviral therapeutics.

The IFN-λ-IFN-λR1-IL-10Rβ Complex Reveals Structural Features Underlying Type III IFN Functional Plasticity

SUMMARY Type III interferons (IFN‐&lgr;s) signal through a heterodimeric receptor complex composed of the IFN‐&lgr;R1 subunit, specific for IFN‐&lgr;s, and interleukin‐10R&bgr; (IL‐10R&bgr;), which is shared by multiple cytokines in the IL‐10 superfamily. Low affinity of IL‐10R&bgr; for cytokines has impeded efforts aimed at crystallizing cytokine‐receptor complexes. We used yeast surface display to engineer a higher‐affinity IFN‐&lgr; variant, H11, which enabled crystallization of the ternary complex. The structure revealed that IL‐10R&bgr; uses a network of tyrosine residues as hydrophobic anchor points to engage IL‐10 family cytokines that present complementary hydrophobic binding patches, explaining its role as both a cross‐reactive but cytokine‐specific receptor. H11 elicited increased anti‐proliferative and antiviral activities in vitro and in vivo. In contrast, engineered higher‐affinity type I IFNs did not increase antiviral potency over wild‐type type I IFNs. Our findings provide insight into cytokine recognition by the IL‐10R family and highlight the plasticity of type III interferon signaling and its therapeutic potential. HIGHLIGHTSThe IFN‐&lgr; ternary complex provides insight into the mechanism of IL‐10R&bgr; engagementHigh‐affinity IFN‐&lgr;3 elicits greater antiproliferative and antiviral activitiesIn vivo, an engineered IFN‐&lgr;3 has enhanced antiviral activity over the wild‐typeIn contrast to IFN‐&lgr;s, high‐affinity type I IFNs do not improve antiviral activity &NA; Using an engineered high‐affinity IFN‐&lgr;, Mendoza et al. solve the structure of the IFN‐&lgr;/IFN‐&lgr;R1/IL‐10R&bgr; ternary signaling complex. The structure reveals how IL‐10R&bgr; can act as both a cross‐reactive but cytokine‐specific receptor. Structure‐activity relationships of engineered type I and III IFNs provide insights into enhancing interferon functional potency.

Liver allografts are toleragenic in rats conditioned with posttransplant total lymphoid irradiation

Background. Posttransplant total lymphoid irradiation (TLI) treatment has been applied to tolerance induction protocols in heart and kidney transplantation models. Methods. We examined the efficacy and mechanism of posttransplant TLI treatment in the induction and maintenance of tolerance in a rat orthotopic liver transplantation model. Results. Posttransplant TLI prolonged ACI (RT1a) liver allograft survival in Lewis (RT1b) hosts, with 50% long-term engraftment without immunosuppression and without evidence of chronic rejection. Injection of donor-type liver mononuclear cells (LMCs) facilitated the prolongation of graft survival, with more than 70% of grafts in LMC recipients surviving more than 100 days without chronic rejection. Recipients with long-term liver allograft survival accepted ACI but not PVG skin grafts. In TLI-conditioned recipients with accepted grafts, apoptosis occurred predominantly in graft-infiltrating leukocytes. In contrast, there were few apoptotic leukocytes in rejecting grafts. Recipients with long-term graft acceptance (>100 days of survival) demonstrated evidence of immune deviation; mixed lymphocyte reaction to ACI stimulator cells was vigorous, but secretion of interferon-&ggr; and interleukin-2 was reduced. In tolerant recipients, the number of Foxp3+ CD25+ CD4+ regulatory T cells was increased in the liver allograft as well as in the peripheral blood. Conclusion. We conclude that posttransplant TLI induces tolerance to liver allografts via a mechanism involving apoptotic cell-deletion and immunoregulation.

Long-term culture of human liver tissue with advanced hepatic functions

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the livers natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.